Cytochrome o is a simple terminal oxidase that has been purified in this laboratory from the primitive procaryote, Vitreoscilla. The cytochrome has an additional enzymatic activity, associated with it at early stages of the purification, which catalyzes the reduction of the cytochrome by NADH to produce, in aerobic solutions, an intermediate, "oxygenated" cytochrome o. The reductase activity has been separated from the cytochrome and purification and characterization of this protein(s) will be continued so that the mechanism of its interaction with cytochrome o can be determined. The possibility that electron transport from NADH to oxygen catalyzed by membrane fragments of Vitreoscilla may be coupled to the synthesis of ATP will be investigated. The eventual goal of these studies is to obtain a soluble bacterial oxidative phosphorylating system with cytochrome o as the terminal acceptor. Membrane fragments will also be used to test for the generation of hydrogen peroxide during the oxidation of NADH to confirm results obtained with the in vitro cytochrome o system. Anaerobic titrations will be performed to determine the number of electrons accepted by cytochrome o from NADH and to determine the midpoint potential of the cytochrome. Rapid kinetic techniques will be employed in collaborative experiments to investigate in detail the electron transfer from NADH to oxygen catalyzed by cytochrome o. Isolated genetic mutants of Vitreoscilla will be studied to see if they contain a cytochrome o with a decreased binding affinity and/or reactivity with oxygen. If successful, studies on the mutant cytochromes will facilitate investigations on the reaction of the cytochrome with oxygen. Investigations on the relationship between heme structure and cytochrome function will be initiated by attempting to reconstitute apocytochrome o with hemes other than protoheme IX. The relative contributions of the protein and the heme moieties to the physiological function of cytochrome o will be ascertainable by the mutant and reconstitution studies. BIBLIOGRAPHIC REFERENCES: Webster, D. A. (1975) The formation of hydrogen peroxide during the oxidation of reduced nicotinamide adenine dinucleotide by cytochrome o from Vitreoscilla. J. Biol. Chem. 250, 4955-4958.